A number of different approaches have been taken to general methods of detection, immobilization, targeting and one-step affinity purification for recombinant proteins. The expression in bacteria of antibody fragments derived from monoclonal antibodies (Skerra, A. & Pluckthun, A., 1988 Science 240 1038-1041; Better, M. et al, 1988 Science 240 1041-1043) or from bacteriophage display repertoires (for review, see Winter, G. et al, (1994) Annual Rev. Immunol. 12 433-435) has reinforced the need for a widely applicable detection system. The initial demonstration that chimeric antibodies could be made where antibodies were fused to other proteins which retained their biological function (M. S. Neuberger & T. H. Rabbitts, 1984; UK patent 2,177,096B) opened the way to the construction of such genetic fusions with antibody molecules.
A variety of fusion tags have been used to date for recombinant proteins: (i) a peptide which recognizes a specific antibody e.g. the myc tag, (Munro, S., and Pelham, H. R. B. (1986) Cell, 46, 291-300; Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T. and Winter, G. (1989) Nature, 341, 544-546); the Flag-peptide (Hopp, T. P. et al (1988) BioTechnology, 6, 1204-1210); the KT3 epitope (Martin, G. A. et al (1990) Cell, 63, 843-849; Martin, G. A., et al (1992) Science, 255, 192-194); an .alpha.-tubulin epitope (Skinner, R. H. et al (1991) J.Biol.Chem., 266, 14163-14166); the T7 gene 10 protein peptide tag (Lutz-Freyermuth, C., Query, C. C. and Keene, J. D. (1990) Proc. Natl. Acad. Sci. U.S.A., 87, 6393-6397) or an affinity support using poly-histidine tails binding to nickel-chelating agarose (Skerra, A., Pfitzinger, I. and Pluckthun, A. (1991) BioTechnology, 9, 273-278; Lilius, G., et al (1991) Eur. J. Biochem., 198, 499-504); the strep-tag, a peptide tag binding to streptavidin (Schmidt, T. G. M. and Skerra, A. (1993) Protein Engineering, 6, 109-122); (ii) a protein domain which forms a complex with a second (macro)molecule e.g. glutathione-S-transferase (Smith, D. B. and Johnson, K. S. (1988) Gene 67, 31-40); bovine pancreatic trypsin inhibitor, BPTI, (Borijin, M. and Nathans, J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 337-341); maltose binding protein, MBP, (Bedouelle, H. & Duplay, P. (1988) Eur. J. Biochem. 171 541-549; Maina, C. V. et al. (1988) Gene 74 365-373), or (iii) a polypeptide sequence that can be biotinylated and thus made to interact with avidin or streptavidin (Schatz, P. J. (1993) Bio/Technology 11, 1138-1143).
Typically, some applications, (such as affinity purification) require a specific but low affinity interaction in order not to impair the function of the recombinant protein with a harsh elution protocol. Other applications (such as targeting and immobilization) require slow release and high-affinity binding of the tag. The known tag systems described above do not have this flexibility and although they perform well in many applications, each of them has some undesired draw-back.
Those tags recognized by specific monoclonal antibodies require columns which are either expensive or difficult to prepare. In some cases, harsh conditions are necessary to elute the bound protein (e.g. extreme pH values or chaotropic agents), which may impair the activity of the purified protein. Moreover, the affinity of the tag for the monoclonal antibody is usually not high enough for some applications, most notably for a stable immobilization of the tagged protein on a biosensor chip, e.g. for use with the BIAcore (Pharmacia).
The latter point applies also to the Strep-tag and perhaps to the polyhistidine tag, although stable immobilization on metal-chelating support might perhaps be achieved by high-density coating of the matrix. The polyhistidine tag has the draw-back that it does not allow protein detection, useful for example in ELISA (enzyme-linked immonusorbent assay) or immunoblot.
When the tag is a protein such as the BPTI tag, protein folding requirements of the tag itself, such as disulfide bridge formation, place limits on the possible applications. Furthermore, the high affinity of BPTI for trypsin (K.sub.d =6.times.10.sup.-14 M), makes elution of BPTI-tagged proteins from trypsin difficult.
Stofko-Hahn et al. ((1992) FEBS Lett. 302, 274-278) have made a fusion of a calmodulin-binding peptide tag derived from the C-terminus of rabbit skeletal muscle myosin light-chain kinase (SK-MLCK) and a recombinant protein and used this in purification on an affinity support.